Two-component signal transduction systems are the primary pathway by which bacteria sense, process and respond to environmental stimuli. The OmpR/EnvZ two-component system has been extensively studied in Escherichia coli, and related enteric bacterium, for its role in the osmoregulation of porin genes. Recent studies in the pathogenic bacterium, Xenorhabdus nematophilus, have led to the discovery of novel features of the OmpR/EnvZ system that remained unrecognized in E. coli. In particular, this system appears to be involved in stationary phase regulation of outer membrane proteins. New evidence obtained from studies in Salmonella typhimurium also suggested that OmpR may play a role in virulence. Based on these recent findings, I propose a new model in which ompR functions to coordinately regulate several gene systems. The global properties of OmpR had not been previously investigated. The hypothesis that OmpR is a multifunctional regulatory protein will be assessed in the enteric bacterium, X. nematophilus. X. nematophilus is a potent insect pathogen that rapidly kills the infected host. At the time of death, free bacteria cannot be detected in the hemolymph of the insect cadaver. It has been speculated that the bacterium survives within the phagocytic hemocytes of the insect hemolymph. In the first phase of this proposal the hemocoelic and cellular location of X. nematophilus will be studied to determine if the bacteria are phagocytized by the insect hemocytes. The well studied Manduca sexta (tobacco horn worm) system will be used to address this question. To assess the multifunctional role of OmpR, ompR null strains of X. nematophilus will be constructed. The proteins produced by the wild-type and ompR strains, grown under relevant environmental conditions, will be compared using one and two dimensional gel electrophoresis. The N-terminal amino acid sequence of OmpR-regulated proteins will be determined. Selected OmpR-regulated genes will be subsequently cloned and sequenced. The ompR strains will be further studied in a variety of in vitro assay systems. Finally, the role of ompR in pathogenicity will be studied in the M. sexta model system using wild-type and ompR strains of X. nematophilus. The distinct advantage of studying ompR function in X. nematophilus is that pathogenic properties of the bacterium can be readily investigated in the laboratory using a natural host. Together, these studies will provide new insights concerning the multifunctional role of OmpR in adaptive responses. Furthermore, the information obtained from these studies in X. nematophilus should further our understanding of the role of OmpR in host-parasite interactions.